Summary: Burkholderia pseudomallei, the etiological agent of melioidosis, is a Gram-negative, facultatively anaerobic, motile bacillus that is responsible for a broad spectrum of illnesses observed in both humans and animals. Burkholderia mallei, the etiological agent of glanders, is a Gram-negative bacterium that is responsible for disease in donkeys, mules, horses and occasionally humans. Unlike the environmental saprophyte B. pseudomallei, however, B. mallei does not persist in nature outside of its soliped hosts. While B. mallei and B. pseudomallei are genotypically similar, significant phenotypic differences do exist between the two pathogenic species. Although glanders is one of the oldest diseases known to man, relatively little is known about the pathogenesis of disease caused by B. mallei. This phenomenon is primarily due to the lack of disease in North America along with the fact that B. mallei can be a particularly dangerous organism to study even in a controlled laboratory environment. Both bacteria are considered BL3 select agents by the CDC. Burkholderia - macrophage interactions: The study of pathogen host cell interactions in vitro is an important tool to define and characterize virulence factors of intracellular bacterial pathogens. The major species of Burkholderia include B. pseudomallei;B. mallei and an avirulent environmentally stable isolate B. thailandensis. B. pseudomallei macrophage interactions have been extensively studied but there is little known about the interactions of B. mallei with macrophages. We have performed a comparative analysis of B. mallei and B. pseudomallei macrophage interactions using the murine macrophage cell line (RAW 264.7). Our findings show that although B. mallei is capable of invading and replicating in RAW cells it is less efficiently internalized and grows more slowly. The optimal multiplicity of infection is critical for permissive B. mallei intracellular growth. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that MOIs significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells. We further tested differences in intracellular survival and multiplication among wild type and various mutants of B. mallei and B. pseudomallei. Eighteen mutants produced in each background of B. mallei and B. pseudomallei were tested in the RAW cell infection model. A type III secretion mutant of B. pseudomallei (strain 26bT3) showed marked differences in internalization and growth in RAW cells. An identical B. mallei type III secretion mutant (BMT3) and a B. mallei LPS mutant (GMrmlD) were incapable of growth in RAW cells. The capsular polysaccharide of B pseudomallei is an essential virulence determinant that is required for replication in murine macrophages, as well as protection from host serum cidal activity and opsonophagocytosis. In a recent study, the immune response directed against a B. pseudomallei capsule mutant (JW270) was investigated in an acute respiratory murine model. JW270 was significantly attenuated in this model (2 log), to levels resembling those of avirulent B. thailandensis. At lethal doses, JW270 colonized lung, liver, and spleen at levels similar to the wild type strain, and was found to trigger a reduced pathology in the liver and spleen. Several cytokine responses were altered in these tissues, and importantly, the levels of IFN- were reduced in the liver and spleen of JW270-infected mice, but not in the lung. These results suggest that the capsular polysaccharide of B. pseudomallei is a critical virulence determinant in respiratory tract infections and that it is an important antigen for generating the Th1 immune response commonly observed in systemic melioidosis. Furthermore, these data suggest that host recognition of B. pseudomallei capsular polysaccharide in the lungs may not be as important to the disease outcome as the innate immune response in the peripheral organs. The results indicated that in vitro and in vivo modeling of virulence using RAW macrophages and mice are a simple and credible approach to screen Burkholderia mutants to beetter understand the pathogenesis glanders and melioidosis. B. mallei is a facultative intracellular pathogen that can cause fatal disease in animals and humans. To better understand the role of phagocytic cells in the control of infections caused by this organism, studies were initiated to examine the interactions of B. mallei with RAW 264.7 murine macrophages. Utilizing modified kanamycin-protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of less than 1. In contrast, the organism was efficiently cleared by the macrophages when infected at an moi of 10. Interestingly, studies demonstrated that the monolayers only produced high levels of TNF-a, IL-6, IL-10, GM-CSF, RANTES and IFN-b when infected at an moi of 10. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that moi significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells. Recent studies have shown that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for survival in a hamster model of glanders. To better understand the role of T6SS-1 in the pathogenesis of disease, studies were initiated to examine the interactions of B. mallei tssE mutants with RAW 264.7 murine macrophages. Utilizing modified gentamicin protection assays, results indicated that although the tssE mutants were able to survive within RAW 264.7 cells, significant replication defects were observed in comparison to controls. In addition, analysis of infected monolayers by DIC microscopy demonstrated that the tssE mutants lacked the ability to induce multinucleated giant cell formation. Via the use of fluorescence microscopy, tssE mutants were shown to undergo escape from LAMP-1 associated vacuoles. Curiously, however, following entry into the cytosol the mutants exhibited actin polymerization defects resulting in inefficient intra- and intercellular spread characteristics. Importantly, all mutant phenotypes observed in this study could be restored by complementation. Based upon these findings, it appears that T6SS-1 plays a critical role in replication and actin-based motility following uptake of B. mallei by RAW 264.7 cells.